V. Amarnath

Cysteine and mercapturate conjugates of oxidized dopamine are in human striatum but only the cysteine conjugate impedes dopamine trafficking in vitro and in vivo

Recent results have suggested that some products of mercapturic acid pathway (MAP) metabolism of oxidized dopamine (DA) may contribute to mesostriatal dopaminergic neurodegeneration, and that at least one product, 5‐S‐cysteinyldopamine (Cys‐DA), is elevated in patients with advanced Parkinsons disease (PD) who have been treated with l‐DOPA. Here we investigated MAP enzymes and products in the midbrain and striatum of control individuals and patients with dementia with Lewy bodies (DLB) who had less severe dopaminergic degeneration than PD patients and who were not treated with l‐DOPA. We also determined the biological activity of MAP metabolites of oxidized DA using primary rat mesencephalic cultures, rat cerebral synaptosomes, and rat striatum in vivo microdialysis. Our results showed that the human mesostriatal dopaminergic pathway generates Cys‐DA but has limited enzymatic capacity for mercapturate formation, that striatal levels of MAP products of oxidized DA are not elevated in DLB patients compared with controls, and that Cys‐DA interferes with trafficking of DA in vitro and in vivo. These results indicate that while Cys‐DA is not increased in striatum of patients with mild dopaminergic neurodegeneration, it may interfere with uptake of DA in patients with advanced PD.

The role of pyrrole formation in the alteration of neurofilament transport induced during exposure to 2,5-hexanedione

Exposure to the -y-diketone, 2,5-hexanedione (HD), results in the accumulation of neurofilaments within the distal axon and is associated with acceleration of neurofilament transport within the proximal axon. The e-amino groups of lysyl residues react with HD forming pyrrole adducts, followed by pyrrole-mediated protein crosslinking. Both reaction steps have been proposed as mechanisms causing neurofilament accumulation and acceleration of transport. In order to assess the importance of these steps on neurofilament transport, we compared transport in the optic system of rats exposed to HD and 3-acetyl-2,5-hexanedione (AcHD), a non-toxic analog of HD which forms pyrroles but does not crosslink proteins. Control, HD-treated, and AcHD-treated rats received intraoptic injections of [33S]-methionine and were exposed to saline, HD, or AcHD by intraperitoneal injections before and during the period of neurofilament transport. Neurofilament triplet proteins in the optic nerve and tract were identified by polyacrylamide gel electrophoresis followed by fluorography. The rate of neurofilament transport was accelerated in HD-treated animals over that of controls. However, despite higher levels of proteinbound pyrroles in AcHD-treated animals, the rate of transport was indistinguishable from that of controls. These findings indicate that pyrrole formation alone is not sufficient to cause acceleration of neurofilament transport.

Comparison of Location, Severity, and Dose Response of Proximal Axonal Lesions Induced by 3,3′-Iminodipropionitrile and Deuterium Substituted Analogs

Administration of 3,3′-iminodipropionitrile (IDPN) to rats results in massive accumulation of tangled neurofilaments in the proximal axons of large neurons, such as in dorsal root ganglia (DRG) and ventral horns of the lumbar spinal cord (LSC). Clinically, rats develop hyperexcitability, circling, head bobbing, and retropulsion. The ultimate toxicant and the molecular mechanism are not known. In a study designed to explore potential activation and detoxification pathways, dose-related differences in location and severity of lesions were observed in rats treated with IDPN or deuterium substituted analogs, 2,2,2′,2′-tetradeuterio-IDPN (2-d-IDPN) or 3,3,3′,3′-tetradeuterio-IDPN (3-d-IDPN). The compounds or saline were administered intraperitoneally to three rats per group at dose levels of 3.0, 1.5, 1.0, and 0.0 mmole/kg/day for 3 days. One week after the initial dose, tissues from DRG and LSC were collected, prepared and evaluated histologically in zones extending from areas adjacent to the cell bodies, distally toward the DRG stalk or toward the lumbar spinal roots. In the low dose IDPN group, DRG and LSC lesions were most prominent in distal zones. As dosage increased, the lesions progressed in severity and in proximity to the cell bodies. At the high dose, lesions were prominent in all zones. The same general pattern occurred with both analogs, although 2-d-IDPN was less potent than IDPN and 3-d-IDPN was more potent than IDPN. The differences in potency from the secondary isotopic effect of deuterium suggest that the 3-position is important in detoxification while the 2-position is important in the bioactivation of IDPN.

Molecular Mechanisms of y-Diketone Neuropathy

We know from human and animal exposure data that inhalation of high concentrations of n-hexane are required, for prolonged periods of time, before a peripheral neuropathy will develop, a product of the rates of production of its ultimate toxic metabolite and of the reactions which occur within the axon (Yamamura, 1969; Herskowitz, et al., 1971). In both human and animal species, the greater vulnerability of long over short axons is a manifestation of the greater number of molecular targets for reaction with the toxic metabolite in the longer axons and a reflection of the stability of those targets. Looking back on the 20 years that we have been aware of this neurotoxicant, it is clear that these conclusions, so important to our understanding of the molecular pathogenesis of n-hexane neuropathy, might have been drawn from the clinical observation of these patients, and, subsequently, of exposed laboratory animals.